Our aim is to understand structure-function relationships in human factor IX. We will use monoclonal antibodies raised against factor IX as site-specific reagents to determine the role of various domains of factor IX in the catalytic and binding functions of the molecule. We will study the kinetics of activation of Gla-domainless factor IX by factor XIa/calcium and by factor VIIa/tissue factor/calcium. To delineate the role of the Gla-domain of factor IXa in its interaction with factor VIIIt, we will compare the abilities of normal and Gla-domainless factor IXa in activating factor X in the absence and presence of cofactors. We are also investigating five abnormal variants of factor IX isolated from unrelated hemophilia-B families. Our initial studies on three of these variants indicate that each variant is defective at or around the active site. Structural and funcitonal studies on the remaining two variants are planned to answer whether the impaired funcitons in these two variants result (1) from failure to undergo rapid activation, or (2) from lack of biologic activity despite normal activation. The latter could stem from an impaired interaction with the cofactors or a defect in the active site. We will examine the availability of the active site in the activated variants by their ability to: a) interact with AT-III, b) activate factor VII, c) activate factor X in the absence of cofactors, and d) incorporate the active site inhibitor dansyl-glu-gly-arg-CK. Interactions of calcium, phospholipid and factor VIIIt will also be investigated. Amino acid sequence studies of the strategic fragments of all five variants are planned to pinpoint precisely the amino acid substitution(s) responsible for the functional defects in these molecules. Using recombinanat DNA techniques, we will obtain nucleotide sequence of the coding regions for each of the variant genes in order to identify the base substitutions responsible for the amino acid replacements. Use of protein chemistry techniques and molecular biology techniques should leave little room for error in sequence studies. Relationship of structural alterations to the functional impairment in variant molecules will contribute to our understanding on a molecular basis of how normal factor IX participates in clotting and why abnormal variants of factor IX fail to function in hemostasis.